She attended Hanyang Girls' High School, where societal expectations for female students were to study literature and marry.

Her decision was influenced by the student protests she witnessed during high school and the tragic death of activist Lee Han-yeol in 1987.

She remembered her love for performing and entertainment, leading her to switch her academic focus to a theater and film major just two months before her college entrance exams, with the goal of studying directing.

[8] After graduating in 1992, Lee began her career as a theater play assistant director, making her debut with the production of A Midsummer Night's Dream in 1991.

Apart from her work in Laundry, Lee collaborated with director Choo Min-joo on reading performances of House with Fig Trees and Scars, as well as the musical Good Morning School.

Costume designer Choi Se-yeon, who was also the producer of the musical Laundry, recommended her for an audition, which led to her landing a minor role in Bong Joon-ho's 2009 film Mother.

[17] In 2013, Lee appeared on television for the first time in The Queen's Classroom and had minor roles in Blue Tower, Royal Villa, and Shining Romance (2013–2014).

[18] In that same year, she won Best Acting at the Seoul Theatre Festival for her role in the play Feast with Hanyang Repertory Theater.

Her performance as the mother who whipped up delicious side dishes for her daughter's prospective in-laws touched many viewers, especially with the line "Please just be pretty to our Seolhee".

Lee was subsequently cast in the supporting role of Ham-an-daek in Mr. Sunshine, the careful and attentive assistant to the character Ae-shin Ae-gi (played by Kim Tae-ri).

Originally envisioned as a minor supporting role, Lee's nuanced performance helped evolve the character into a more prominent maternal figure, filling the absence of the female lead's parents.

She played the supporting character of Gook Moon-gwang, the housekeeper of Park family, who also worked for the house's architect and previous owner.

[25] She and her co-stars made history by winning Best Ensemble at the Screen Actors Guild Awards for a non-English language film.

In this drama, Lee showcased her range by delivering thrilling and emotional moments as the mother of the lead character, Dong-baek.

In 2020, Lee starred as Kang Cho-yeon in the KBS2 family weekend drama series Once Again, which aired from March 28 to September 13, 2020, every Saturday and Sunday.

[27][28] In that same year, Lee Jung-eun starred in Park Ji-wan's directorial debut film The Day I Died: Unclosed Case as Suncheondaek, along with Kim Hye-soo.

Based on a true story, the film features historical materials meticulously researched by director Lee Joon-ik.

Directed by Hong Jong-chan, the series focuses on characters at a district juvenile court handling cases involving young offenders.

Lee's character clashes with elite judge Shim Eun-seok (Kim Hye-soo) due to their differing approaches to the cases.

The jury selected Lee for this award based on her portrayal of desire, frustration, and the courage displayed by a middle-aged woman whose dreams were delicately and realistically depicted in Hommage.

[36] In the end of 2022, Lee appeared in Missing: The Other Side Season 2 as Kang Eun-sil, a captain with 30 years of experience who now resides in a mysterious village inhabited by spirits.

[41][42] In 2023, Lee joined the cast of Netflix's original series Daily Dose of Sunshine as Song Hyo-shin, the chief nurse of the Psychiatric Department.